docs/protocols/rip_qpcr_protocol_v1.yaml

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# Protocol metadata
id: PROT-0014
name: RNA Immunoprecipitation qPCR Protocol
version: 1.0
description: Protocol for RNA immunoprecipitation followed by RT-qPCR to detect RNA-protein interactions
author: Jordan Lab
created: 2025-05-06
last_updated: 2025-05-07
category: molecular-biology

# Materials required
materials:
  - name: Cell culture dishes
    type: 10-cm or 15-cm dishes
    notes: Depending on protein/RNA abundance
  - name: PBS (cold)
    temperature: 4°C
    storage: Room temperature
  - name: UV crosslinker
    wavelength: 254 nm
    alternative: Formaldehyde crosslinking
  - name: Lysis buffer
    components: "Non-denaturing buffer with RNase inhibitors"
    storage: Prepare fresh or store at -20°C
  - name: Protein A/G beads
    preparation: Pre-blocked with BSA and tRNA
    storage: 4°C
  - name: Antibodies for target protein
    dilution: As recommended for IP
    storage: -20°C
  - name: IgG control antibody
    use: Negative control
    storage: -20°C
  - name: Glycine solution
    concentration: 2.5 M (for formaldehyde quenching)
    storage: Room temperature
  - name: RNA isolation reagents
    type: TRIzol or column-based kit
    storage: According to manufacturer's instructions
  - name: RT-qPCR reagents
    components: "Reverse transcription kit, qPCR master mix, primers"
    storage: -20°C

# Equipment required
equipment:
  - name: UV crosslinker
    settings: 150-300 mJ/cm²
    alternative: Formaldehyde crosslinking setup
  - name: Refrigerated centrifuge
    settings: Various speeds, 4°C
  - name: Rotating mixer
    temperature: 4°C
  - name: Thermocycler
    use: RT-qPCR and crosslink reversal
  - name: qPCR instrument
    use: Target RNA detection

# Protocol steps - General workflow
steps:
  - step: 1
    action: "Grow cells to desired confluency"
    details: "80-90% confluency recommended for optimal yield"
  - step: 2
    action: "Perform crosslinking"
    details: "Choose between UV or formaldehyde crosslinking methods (see detailed workflows below)"
  - step: 3
    action: "Harvest and lyse cells"
    details: "Use buffer with RNase inhibitors; keep samples cold"
  - step: 4
    action: "Pre-clear lysates"
    details: "Incubate with protein A/G beads alone for 1 hour at 4°C to reduce nonspecific binding"
  - step: 5
    action: "Perform immunoprecipitation"
    details: "Add specific antibody, incubate, then add protein A/G beads"
  - step: 6
    action: "Wash beads"
    details: "Multiple washes with increasing stringency to remove non-specific binding"
  - step: 7
    action: "Reverse crosslinks if needed"
    details: "UV: proteinase K digestion; formaldehyde: heat treatment (65°C)"
  - step: 8
    action: "Isolate RNA"
    details: "Extract RNA from immunoprecipitated complex using TRIzol or column-based kit"
  - step: 9
    action: "Perform RT-qPCR"
    details: "Reverse transcribe RNA and quantify target RNA enrichment"

# UV crosslinking workflow
uv_crosslinking_workflow:
  - step: 1
    action: "Grow cells to desired confluency in culture dishes"
    details: "Use 10-15 cm dishes for sufficient material"
  - step: 2
    action: "Wash cells with cold PBS"
    details: "Remove media and serum proteins; keep cells on ice to minimize RNase activity"
  - step: 3
    action: "Add fresh cold PBS"
    details: "Add enough to cover the cells but minimize UV light absorption"
  - step: 4
    action: "Perform UV crosslinking"
    details: "Use UV 254 nm at 150-300 mJ/cm²; optimize for your protein of interest"
  - step: 5
    action: "Harvest cells"
    details: "Scrape or gently trypsinize cells and collect by centrifugation"
  - step: 6
    action: "Lyse cells"
    details: "Use mild conditions suitable for maintaining RNP complexes"
  - step: 7
    action: "Pre-clear lysates"
    details: "Incubate with protein A/G beads alone to reduce nonspecific binding"
  - step: 8
    action: "Perform immunoprecipitation"
    details: "Add the specific antibody, followed by protein A/G beads"
  - step: 9
    action: "Wash beads"
    details: "Multiple washes with increasing stringency"
  - step: 10
    action: "Digest and extract RNA"
    details: "Treat with proteinase K and extract RNA"
  - step: 11
    action: "Perform RT-qPCR"
    details: "Reverse transcribe and perform qPCR to detect target RNAs"

# Formaldehyde crosslinking workflow
formaldehyde_crosslinking_workflow:
  - step: 1
    action: "Grow cells to desired confluency"
    details: "Use 10-15 cm dishes for sufficient material"
  - step: 2
    action: "Prepare formaldehyde solution"
    details: "Prepare fresh formaldehyde at working concentration (often 1% final in culture medium)"
  - step: 3
    action: "Crosslink cells"
    details: "Add formaldehyde directly to cells and incubate for 5–10 minutes at RT or 37°C"
  - step: 4
    action: "Quench reaction"
    details: "Add glycine (125 mM final) for 5–10 minutes to quench formaldehyde"
  - step: 5
    action: "Wash cells"
    details: "Wash thoroughly with cold PBS to remove formaldehyde"
  - step: 6
    action: "Harvest cells"
    details: "Scrape cells carefully and collect by centrifugation"
  - step: 7
    action: "Lyse cells"
    details: "Use conditions that preserve protein-RNA complexes"
  - step: 8
    action: "Perform immunoprecipitation"
    details: "Use specific antibody against protein of interest"
  - step: 9
    action: "Wash beads"
    details: "Multiple washes to remove nonspecific material"
  - step: 10
    action: "Reverse crosslink"
    details: "Heat at 65°C for several hours with SDS/high salt"
  - step: 11
    action: "Isolate RNA"
    details: "Extract RNA from immunoprecipitated sample"
  - step: 12
    action: "Perform RT-qPCR"
    details: "Reverse transcribe and quantify targets by qPCR"

# Critical parameters
critical_parameters:
  - parameter: "Crosslinking method selection"
    details: "UV is more specific for direct interactions; formaldehyde captures larger complexes"
  - parameter: "Antibody specificity"
    details: "Validate antibody IP efficiency by Western blot before RIP"
  - parameter: "RNase control"
    details: "Use RNase inhibitors in all buffers and work quickly at 4°C"
  - parameter: "Wash stringency"
    details: "Balance between removing background and maintaining specific interactions"

# Troubleshooting
troubleshooting:
  - problem: "Poor RNA yield"
    solution: "Increase starting material; optimize crosslinking conditions; check for RNase contamination"
  - problem: "High background in control IPs"
    solution: "Increase wash stringency; pre-block beads with BSA/tRNA; use more specific antibody"
  - problem: "No enrichment of target RNA"
    solution: "Verify protein-RNA interaction using alternative approach; check crosslinking efficiency; try different antibody"

# Method comparisons
considerations:
  uv_crosslinking:
    - advantage: "Standardize irradiation distance/energy for reproducibility"
    - advantage: "More specific crosslinks between directly interacting residues"
    - advantage: "Better for pinpointing precise binding sites"
    - disadvantage: "Requires specialized equipment (UV crosslinker)"
    - disadvantage: "May damage RNA integrity at high doses"
    - note: "More common in CLIP-based methods for precise interaction mapping"
  formaldehyde_crosslinking:
    - advantage: "Straightforward chemical method without specialized equipment"
    - advantage: "Captures indirect interactions within larger complexes"
    - advantage: "Often higher yield"
    - disadvantage: "Higher background from nonspecific crosslinks"
    - disadvantage: "Reversal requires potentially harsh conditions (heating, high salt)"
    - note: "Better for detecting weak or transient interactions"

# Safety considerations
safety:
  ppe: "Lab coat, gloves, and eye protection required"
  hazards: "UV radiation: avoid direct exposure; Formaldehyde: toxic and carcinogenic, use in fume hood"
  waste: "Dispose of crosslinking reagents according to institutional guidelines"

# Quality control
quality_control:
  - check: "Include IgG control IP"
    criteria: "Should show minimal enrichment of target RNAs"
  - check: "Include input RNA sample"
    criteria: "For normalization and calculating percent input"
  - check: "Test known RNA-protein interaction"
    criteria: "Should show significant enrichment over IgG control"

# Data analysis
data_analysis:
  - step: 1
    action: "Calculate fold enrichment"
    details: "Compare target RNA in specific IP vs. IgG control IP"
  - step: 2
    action: "Calculate percent input"
    details: "Compare RNA abundance in IP vs. a defined percentage of input material"
  - step: 3
    action: "Statistical analysis"
    details: "Perform appropriate statistical tests on biological replicates"

# Best practices
best_practices:
  - "Run pilot experiments to optimize crosslinking conditions for specificity vs. yield"
  - "Test different crosslinking strengths and confirm with a known positive RNA target"
  - "Include IgG control or nonspecific antibody to measure background binding"
  - "Use input RNA to normalize or calculate percentage of input in qPCR"
  - "If possible, use a known RNA-protein interaction as positive control"
  - "Incorporate RNase inhibitors in all buffers and keep samples cold"
  - "For many RIP-qPCR experiments, mild UV crosslinking at 254 nm is preferred"

# References
references:
  - "Peritz T, et al. (2006) Immunoprecipitation of mRNA-protein complexes. Nat Protoc. 1(2):577-580"
  - "Keene JD, et al. (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc. 1(1):302-307"
  - "Niranjanakumari S, et al. (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods. 26(2):182-190"

# Notes
notes: |
  - Two crosslinking methods are described: UV (254 nm) and formaldehyde
  - UV crosslinking is more specific but requires specialized equipment
  - Formaldehyde crosslinking is simpler but may have higher background
  - Always include appropriate controls (IgG, input RNA)
  - Consider CLIP-based methods for more precise mapping of binding sites
  - For studying RNA binding proteins that interact with many targets, consider combining with RNA-seq (RIP-seq)
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